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Development of a Cross-Reactive MonoclonalAntibody to SulfonamideAntibiotics: Evidence forStructural Conformation-Selective Hapten RecognitionMARK T. MULDOON , IVAN A. FONT , ROSS C.BEIER , CAROL K. HOLTZAPPLE , COLIN R. YOUNG& LARRY H. STANKERPublished online: 01 Jul 2010.

To cite this article: MARK T. MULDOON , IVAN A. FONT , ROSS C. BEIER , CAROLK. HOLTZAPPLE , COLIN R. YOUNG & LARRY H. STANKER (1999): Development ofa Cross-Reactive Monoclonal Antibody to Sulfonamide Antibiotics: Evidence forStructural Conformation-Selective Hapten Recognition, Food and AgriculturalImmunology, 11:2, 117-134

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ISSN 0954 ±0105 (print)/ISSN 1465±3443 (online)/99/020117±18 � 1999 Taylor & Francis Ltd

Food and Agricultural Immunology (1999) 11, 117±134

Correspondence to: R. C. Beier. Tel: + 1 409 260 9411; Fax: + 1 409 260 9332;E-mail: RCBeier@ffsru.tamu.eduMention of a trade name, proprietary product, or specific equipment does not constitute a guarantee orwarranty by the US Department of Agriculture and does not imply its approval to the exclusion of otherproducts that may be suitable.

Development of a C ross-Reactive Monoclonal Antibody toSulfonamide Antibiotics: Evidence for Structural

Conformation-Selective Hapten Recognition

MARK T. MULDOON, IVAN A. FONT, ROSS C. BEIER,CAROL K. HOLTZAPPLE, COLIN R. YOUNG A N D LARRY H. STANKER

Food Animal Protection Research Laboratory, Agricultural Research Service, USDepartment of Agriculture, 2881 F & B Road, College Station, TX 77845 ± 4998, USA

(Original manuscript received 20 November 1998; revised manuscript accepted 24 February 1999)

A unique anti-sulfonamide antibody-secreting hybridoma that cross-reacts with severalsulfonamides was isolated. This was possible by using a N-sulfanilyl-4-aminobenzoic acid

hapten ± protein conjugate as the immunogen. Most of the antibodies that were detected in

immunized mice did not recognize the free drug. However, by screening a large number ofantibody-secreting hybridomas, cell lines were isolated that produced antibodies which

recognized the free drug. The sensitivities of one such monoclonal antibody (MAb), referredto as Sulfa-1, for sulfanitran, sulfapyridine and sulfathiazole (expressed as IC50 values), were

1.41, 22.8, and 322 ng ml ± 1, respectively. Molecular modeling studies showed that the

calculated minimum energy conformation of the hapten used as immunogen was differentthan those of the cross-reactive drugs. It was postulated that the MAb was derived from a cell

line responsive to a form of the immunogen in which the structural conformation of thehapten was different than the molecular modeling calculated minimum energy conformation

of the hapten. This was supported by further molecular modeling studies, including the use

of potential energy-conformational maps, and competitive ELISA experiments conducted attwo different temperatures. The immunogen appeared to be in a structural conformation

achieved at an energy of + 0.193 kcal mol ± 1 with an energy barrier of 3.13 kcal above theminimum energy conformation; an energy easily found within the body temperature of the

immunized mouse. Design of haptens for the purpose of generating cross-reactive antibodies

should not just consider the two-dimensional structure, but also the three-dimensionalconformation as well as the various structural combinations that can be easily attained

within the body temperature of the immunized animal.

Keywords: Antibiotics, ELISA, hapten recognition, molecular modeling, monoclonal

antibody, protein conjugate, sulfanitran, sulfapyridine, sulfathiazole, sulfonamides

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118 M. T. MULDOON ET AL.

INTRODUCTION

The sulfonamide antibiotics are widely used to treat bacterial and protozoan infections in

food animals, and as a result, residues have appeared in food products (Franco et al., 1990).

Currently in the USA, the tolerance level for sulfonamides in animal-based food products is

0.1 m g g±1 (USDA, 1994). Conventional residue methods for sulfonamides include bioassays,

thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC)

(Aerts et al., 1995). These methods are often labor intensive, non-specific, or lack the

necessary detection limits for a suitable residue monitoring method. Therefore, ELISAs were

developed for several of the sulfonamides (e.g. Fleeker & Lovett, 1985; Dixon-Holland &

Katz, 1988; Jackman et al., 1993). Polyclonal antibodies (PAbs) were generated for these

methods using immunogens in which the sulfonamide of interest was linked to a carrier

protein via the N 4-position (see Table 1). In doing so, N 1-substituents, which are specific for

individual sulfonamides were readily exposed to the immune system and this resulted in the

production of antibodies which were highly specific for the particular sulfonamide drug used

as the hapten. To date, few monoclonal antibody (MAb)-based immunoassays for

sulfonamide antibiotics have been reported in the literature (M Èartlbauer et al., 1994).

We are interested in developing a sensitive, class-specific immunoassay which can be used

to detect several sulfonamide drugs, and therefore reduce the number of analyses required to

characterize food samples for sulfonamide contamination. This was the focus of a previous

study in which a sulfathiazole (Table 1) derivative was linked to a carrier protein via the

N 1-thiazolyl ring system and used as the immunogen to produce PAbs (Sheth & Sporns,

1991). These antibodies cross-reacted with several sulfonamide drugs, however the ELISAs

were not sensitive (1±25 m g ml±1 ).

In the approach described here, a structurally different sulfonamide hapten was

synthesized and conjugated to a carrier protein via the N 1-sulfonamide ring system for use

as the immunogen. It was hypothesized that the antibodies produced from this hapten should

be specific to the p-aminobenzoyl ring moiety, which is common to all sulfonamides, and

therefore may bind many of the sulfonamide drugs. A rare MAb was isolated from

immunized mice which reacted with several important sulfonamides. In addition,

significantly improved detection limits were observed for various cross-reactive sulfona-

mides other than those previously described. Molecular modeling studies of the hapten and

sulfonamide drugs were conducted in order to describe potential sites of antibody ±

sulfonamide recognition. The results suggest that antibody ±sulfonamide recognition is

governed in part by conformational characteristics of the molecule. These results should aid

in the design of future sulfonamide hapten±protein conjugates.

MATERIALS AND METHODS

C hemicals and SuppliesStructures of the sulfonamides used in this study are illustrated in Table 1. Sulfathiazole,

sulfisomidine, sulfamethazine, sulfisoxazole, 4-acetylsulfanilyl chloride, 4-aminobenzoic

acid and 2-aminopyridine were purchased from Aldrich (Milwaukee, WI, USA). Sulfadime-

thoxine, sulfaquinoxaline, and pyridine were purchased from Fluka (Buchs, Switzerland).

Sulfamerazine and sulfanilic acid were purchased from Lancaster Synthesis (Windham, NH,

USA). Sulfapyridine, sulfadiazine, sulfacetamide, sulfanilamide, sulfamethoxazole, sulfame-

thizole, sulfabenzamide, sulfachloropyridazine, sulfanitran, goat anti-mouse IgG (whole

molecule) conjugated to horseradish peroxidase (G a MIgG ±HRP), and p-nitrophenyl

phosphate tablets were purchased from Sigma (St Louis, MO, USA). Sulfasalazine and

N 4-acetylsulfamethazine were purchased from ICN Biomedicals (Costa Mesa, CA, USA).

N 4-Acetylsulfanilamide was purchased from Chem Service (West Chester, PA, USA).

OmniSolv grade acetone and diethyl ether were from EM Science (Gibbstown, NJ, USA).

Methylene chloride (B&J grade) was from Baxter (McGaw Hill, IL, USA). Keyhole limpet

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 119

TABLE 1. Structures of the various sulfonamides used in this studya

aN 4-acetyl derivatives of sulfapyridine, sulfamethazine, and sulfanilamide are not shown.

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120 M. T. MULDOON ET AL.

hemocyanin (KLH) was purchased from Pierce (Rockford, IL, USA). Bovine serum albumin

(fraction V, 98%) (BSA) was obtained from Hazelton Biologics (Lenexa, KS, USA). K-Blue

(enzyme substrate) was purchased from ELISA Technologies (Lexington, KY, USA). RIBI

adjuvant R-700 was from RIBI ImmunoChem Research, Inc. (Hamilton, MT, USA). Mouse

antibody isotyping kit was purchased from Southern Biotechnology Associates, Inc.

(Birmingham, AL, USA). Non-fat dry milk (NFDM) was obtained from a local grocery

store.

BuffersAll buffer components were cell culture or reagent grade. Assay buffer (pH 7.75) contained

per liter of water: 11.4 g Tris±HCl, 3.32 g Tris base, 8.7 g sodium chloride, 0.01 g NFDM, and

0.005% (v/v) Tween-20. Coating buffer (pH 9.6) contained per liter of water: 1.59 g sodium

carbonate, 2.93 g sodium bicarbonate and 0.203 g magnesium chloride. Phosphate-buffered

saline (pH 7) (PBS-7) contained per liter of water: 8.0 g sodium chloride, 0.2 g potassium

chloride, 1.15 g sodium phosphate (dibasic), and 0.2 g potassium phosphate. Blocking buffer

(pH 9) contained 30 g NFDM per liter of PBS.

Equipm entCell culture plasticware was obtained from Costar (Cambridge, MA, USA). Microtiter plates

used for ELISAs were flat bottom Nunc Immunoplate II Maxisorp (Nunc, Roskilde,

Denmark). Microtiter plate optical density (OD) measurements were made using a Bio-Rad

Model 3550 microplate reader (Richm ond, CA, USA). Data were collected using a

Macintosh II computer and Reader Driver 1.0 and Microplate Manager 1.0 software (Bio-

Rad). Other calculations utilized Excel spreadsheet software (Microsoft Corp., Redmond,

WA, USA).

Proton nuclear magnetic resonance spectrometry (1H-NMR) was carried out on a Varian

Unity-Plus 500 instrument (500 MHz) (Fernando, CA, USA) at the NMR Laboratory

Services, Department of Chemistry, Texas A&M University, College Station, Texas, USA.

Positive ion fast atom bombardment mass spectrometry ( + FAB/MS) was carried out on a

VG Analytical 70S high resolution, double focusing, magnetic sector instrument (Man-

chester, UK) at the Mass Spectrometry Facility, Department of Chemistry, Texas A&M

University (NSF Grant CHE-870569 7).

Hapten Synthesis

N-(4-Acetylsulfanilyl)-4-aminobenzoic acid (I). The method used for synthesis was adapted

from Winterbottom (1940). 4-Acetylsulfanilyl chloride (10 g, 42.8 mmol) and 4-amino-

benzoic acid (5.84 g, 42.5 mmol) were dissolved in acetone (40 ml) containing 8% (v/v)

pyridine. The orange colored solution was stirred overnight. The acetone was evaporated and

distilled water (120 ml) was added to the orange oil. The orange precipitate was filtered,

rinsed with cold ethanol (10 ml) and transferred to a beaker containing hot water (200 ml).

Ethanol (75 ml) was added to dissolve the solid. After evaporating part of the ethanol by

heating, the clear solution was left standing at room temperature overnight. The precipitate

was filtered, washed successively with distilled water (100 ml), cold 95% ethanol (20 ml) and

diethyl ether (40 ml) and air dried to obtain 12.17 g (85.6% recovery) of a cream colored

solid.

Deacetylation of I to N-sulfanilyl-4-aminobenzoic acid (SUL). Product I (5.0 g, 15 mmol)

was refluxed in 0.5 M -NaOH (50 ml) overnight. The solution was cooled in an ice bath and

acidified to pH 6.0 with concentrated HCl. It was extracted two times with methylene

chloride (100 ml) and the organic phase dried over anhydrous sodium sulfate. The extract was

filtered and evaporated to dryness affording 1.5 g (34% recovery) of a tan solid. 1H-NMR

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 121

(500 MHz, DMSO-d6 ): d 12.61 (br s, 1 H COOH ), 10.42 (s, 1 H, NH ), 7.77 (d, 2 H, 8.7 ArH ),

7.44 (d, 2 H, 8.7, ArH ), 7.14 (d, 2 H, 8.7, ArH ), 6.54 (d, 2 H, 8.7 ArH ), 6.05 (br s, 2 H, NH ).

+ FAB/MS (M + 1), m/z 293.

Synthesis of N4-acetylsulfapyridine (N4-ASPY). The method was adapted from Winter-

bottom (1940). 4-Acetylsulfanilyl chloride (10 g, 43 mmol) and 2-aminopyridine (4 g,

50 mmol) were dissolved in acetone (40 ml) containing 8% (v/v) pyridine and the mixture

was left standing overnight at room temperature. The yellow reaction mixture was cooled to

4ÊC for 48 h. The crystalline precipitate was collected and washed successively with cold

water (200 ml), cold acetone (300 ml), water (100 ml), and finally acetone (300 ml) affording

4.08 g (34% recovery) of a white crystalline solid. 1H-NMR (500 MHz, DMSO-d6 ): d 11.80

(very br s, 1 H, NH ), 10.70 (br s, 1 H, NH ), 8.01 (d, 1 H, 4.4, ArH ), 7.79 (d, 2 H, 8.7, ArH ),

7.69 (br m, 3 H, ArH ), 7.11 (d, 1 H, 8.7, ArH ), 6.86 (t, 1 H, 6.2, ArH ), 2.06 (s, 3 H, CH3 ).

+ FAB/MS (M + 1), m/z 292.

Protein C onjugationThe procedures used for hapten±protein conjugations were adapted from Sheth & Sporns

(1991). SUL (62.5 mg), NHS (34.5 mg), and DCC (45.3 mg) were com bined in DMF (1.5 ml).

The mixture was stirred for 18 h at 4ÊC. The white precipitate was removed by filtration and the

liquid (containing the NHS ester of SUL) was used for subsequent reactions. For KLH

conjugation (KLH-SUL), 0.23 ml of the NHS ester of SUL was reacted with KLH (10 mg) in

deionized water (2 ml). DMF (1.8 ml) was added to dissolve the precipitate, the pH was

adjusted to 7.6, and the solution was stirred overnight. For BSA conjugation (BSA ±SUL),

0.23 ml of the NHS ester of SUL was added to BSA (55 mg) in PBS-7 (2.0 ml). The pH was

adjusted to 7.6 and the solution was stirred for 24 h at room temperature. Both of the conjugates

were dialyzed against 50% (v/v) PBS-7 containing 20% (v/v) DMF, 50% (v/v) PBS-7 (four

times), and the finally deionized water. The conjugates (in water) were stored at ±20ÊC.

Direct Binding ELISAAssay plates were coated with BSA ±SUL (100 ng) in 100 m l coating buffer and incubated

overnight at 4ÊC. The plates were exhaustively washed with distilled water containing 0.05%

Tween-20 followed by a distilled water rinse. The plates were incubated with blocking buffer

(200 m l) for 1 h at room temperature and then washed. The plates were either used

immediately or frozen at ±20ÊC until used. For the assay, aliquots of cell culture supernatant

(100 m l) were added to wells of an assay plate, incubated for 1 h at room temperature, and

washed. Then, G a MIgG ±HRP diluted 1:500 in assay buffer (100 m l) was added to each well,

incubated 1 h, and the plate was washed. Enzyme substrate (100 m l) was added to each well

and optical density measurements (655 nm) of the plate were taken after 30 min. The enzym e

product color development was visually monitored during screening of fusion plates.

C ompetitive Inhibition ELISA (cELISA)Microtiter plates were coated and blocked as previously described. Competitors diluted in

assay buffer (100 m l) were added to the microtiter plate well followed by antibody diluted

in assay buffer (100 m l). The amount of antibody used was that amount which produced

a minimum optical density reading of 0.7 absorbance units and was in the linear portion

of the titration curve as determined using the direct ELISA. The sample antibody mixture

was incubated at room temperature for 1 h and then the plate was washed. Then,

G a ±MIgG-HRP diluted 1:500 in assay buffer (100 m l) was added, incubated at room

temperature for 1 h, and then the plate was washed. Enzym e substrate (100 m l) was added

and plate optical density measurements (655 nm ) were taken at 30 min. IC50 values

(concentration of inhibitor which produces a 50% decrease in the signal of the no

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122 M. T. MULDOON ET AL.

competitor control) for the various sulfonamides were obtained using the 4-parameter

curve fitting function in Microplate Manager 1.0.

MAb ProductionFive BALB/c mice were injected intraperitoneally (i.p.) and intramuscularly (i.m.) with

100 m l and 50 m l, respectively, of a solution containing 25 mg/ml±1 KLH ±SUL in RIBI

adjuvant. The mice were boosted 21 days later with 100 m l (i.p.) of KLH ±SUL in RIBI

adjuvant. On day 30, the mice were bled (tail) and the sera was collected, titrated and tested

using a cELISA and a panel of selected sulphonamides (SUL (hapten), SPY, SDZ, SMZ,

STZ, SAA, SAM, and SBM (Table 1). On day 41, 4 days prior to the cell fusion, one mouse

received 100 m l of a 1.2 mg ml±1 solution of BSA ±SUL in distilled water in the tail vein. The

spleen was removed and splenocytes were fused with SP2/O myeloma cells and cultured in

96 well plates using conditions previously described (Stanker et al., 1986).

Hybridoma ScreeningTissue culture fluid was screened for antibody-producing hybridomas using a direct binding

ELISA. Hybridomas from wells which showed a strong positive response in the initial

screening were expanded and rescreened using a cELISA format. The com petitors were SUL,

SPY, SDZ, SMZ, STZ, and SDM (Table 1) at a single concentration of 1 m g ml±1 .

Hybridomas which demonstrated inhibition of antibody binding by free drug were subcloned

twice by limiting dilution to ensure monoclonal origin. Isotyping was performed using the

direct binding ELISA in combination with antimouse isotype antibodies conjugated to

alkaline phosphatase.

C haracterization of MAb Sulfa-1 for Sulfonamide RecognitionMAb Sulfa-1 was characterized for reactivity toward the sulfonamides listed in Table 1. This

was performed using the cELISA format. Typically, on each plate, nine concentrations of

SPY (50 to 0.19 ng/well) were analyzed in duplicate and nine concentrations of 2 other

sulfonamides (1000 to 3.91 ng/well) were analyzed in triplicate. The experiment was repeated

on a separate day. Higher concentrations were tested for those sulfonamides which did not

show inhibition in the first experiment. IC50 values were obtained as previously described.

Kinetic ELISA ExperimentscELISAs were performed using SPY and SNT as the competitors. The cELISA protocol was

conducted as previously described except for the competition step, in which the antibody was

incubated with the sample (with or without com petitor) on the BSA ±SUL-coated plate at

either room temperature (approximately 22ÊC) or 37ÊC for either 0.5, 1, 2 or 6 h. All other

steps were the same as the cELISA.

Molecular Modeling Studies

Determination of minimum energy conformations. Molecular modeling studies were

conducted using a CAChe WorkSystem operated on a Macintosh Quadra 700 computer

equipped with a RP88 coprocessor board and a stereoscopic display (CAChe Scientific, Inc.,

Beaverton, OR, USA). Minimum energy conformations of the various structures were

calculated using a modification of Allinger’ s standard MM2 force field parameters (Allinger,

1977). Initial conformation optimization was followed by a sequential search for low energy

conformations in which all of the dihedral angles of the compound were rotated 360Ê in 15Ê

increments using molecular mechanics. Multiple low energy conformations were selected

and re-optimized for further energy mimimization. Potential energy conformation maps were

constructed using the lowest minimum energy conformation thus obtained. These were

calculated by re-evaluating the minimal energy conformation in an exhaustive search using

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 123

molecular mechanics in which two of the dihedral angles (indicated in Figure 3) were rotated

independently 360Ê in 15Ê increments.

Determination of electronic properties. The electronic wavefunction was calculated for

some compounds utilizing Extended H Èuckel (Hoffmann, 1963). The CAChe Tabulator

application (CAChe Scientific, Beaverton, OR, USA) was used to convert these data into

three-dimensional coordinates in order to visualize electron density and electrostatic

potential. The value used for the electron density probability was 0.01 electrons ‹±3.

RESULTS AND DISCUSSION

Antibody ProductionFive mice were immunized with KLH-SUL (See Table 1 for sulfonamide structures).

Analysis of the sera from these mice, showed that three had relatively high antibody titers on

immobilized BSA-SUL (Figure 1). Antibody titers on plates treated with BSA alone were

significantly lower for all the test sera. Sera obtained from non-immunized mice did not show

significant binding .

Immune sera were tested in a cELISA to determine if antibody binding to BSA ±SUL

could be inhibited by free sulfonamide. Table 2 shows the results from this experiment.

Serum from mouse 1 had a high antibody titer and competition with free hapten and

several of the sulfonamide drugs. Mice 2 and 4 showed low antibody titers and

competition with only free hapten. Mouse 3 had a high antibody titer and com petition with

only free hapten. Mouse 5 had a high antibody titer but did not show competition with

the unconjugated hapten (SUL) or SMZ, suggesting that the majority of the antibodies

produced by this mouse recognized the conjugated form of the hapten or the linkage

chemistry since it did not bind BSA alone at the sera dilution used (Figure 1). Four out

of the five mice that were immunized produced antibodies that recognized the free hapten

but only one of these recognized free sulfonamide drug.

For mouse 1, the sensitivities for the cross-reactive drugs (Table 2) were comparable to the

values reported previously for cross-reactive anti-sulfonamide rabbit polyclonal antibodies

FIG. 1. Titration curves for sera from KLH ±SUL-immunized mice on BSA ±SUL-coated plates (left panel)and BSA-coated plates (right panel) using a direct binding ELISA. j , Mouse 1; d , Mouse 2;m , Mouse 3; . , Mouse 4; r , Mouse 5; + pre-immunised sera.

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124 M. T. MULDOON ET AL.

raised against a different N 1-linked hapten-protein conjugate (Sheth & Sporns, 1991) .

Although the data are limited, PAb sensitivity toward sulfonamide drugs using these types of

conjugates may be limited to this range of sensitivity due to the presence in the serum of a

large amount of antibodies that bind immobilized hapten but are not inhibited by free hapten.

Mouse 1 was selected as a source of splenocytes for cell fusion since its serum showed a

relatively high antibody titer and more importantly, the antibodies cross-reacted with several

of the sulfonamide drugs tested.

Hybridoma Screening and MAb ProductionTen days following cell fusion, approximately 90% of the wells of the thirty 96-well

microculture plates contained 1±5 hybridom a colonies. Supernatants from these plates were

screened for the presence of anti-sulfonamide antibodies using a direct binding ELISA.

Positive wells (138) were visually selected and the hybridoma cells from these were

expanded into 24-well microculture plates. Three days later they were screened in a cELISA

format using free hapten and five different sulfonamide drugs (SPY, SDZ, SMZ, STZ, and

SDM). The results of this second screen indicated that 86 of the original 138 positives

contained hybridomas that continued to produce detectable antibody. Of these, antibody

binding was not inhibited with free hapten or any of the five sulfonamide drugs in 75 of the

cases (86.2%). In three cases (3.4%) antibody binding was inhibited by free hapten but not

by a sulfonamide drug. Six of the wells (6.5%) produced antibody that was inhibited by free

hapten and SPY, and finally, two wells (2.3%) produced antibody that was inhibited by free

hapten, SPY, and STZ. One of the two hybridom as in this last group was subcloned for

further study.

These results suggest that the percentage of cross-reactive antibody-producing splenocytes

in the total population that were capable of fusing with myeloma cells was low. However,

since cross-reactive antibodies were detected in the serum of Mouse 1, the population of

plasma cells producing cross-reacting antibodies may have been elevated in this animal. In

contrast, the most prevalent antibodies produced by the other four immunized mice were not

inhibited by free hapten or free drug. These data suggest that antibodies capable of binding

a broad range of sulfonamide drugs represent a rare population in these animals using KLH ±

SUL as the immunogen.

C haracterization of MAb Sulfa-1 for Sulfonamide RecognitionMAb Sulfa-1 was identified as an IgG 2a ( k light chain) and characterized for cross-reactivity

toward the free hapten and a panel of sulfonamide drugs. Typical competitive inhibition

TABLE 2. Cross-reactivity of sera from immunized mice with selected sulfonamides

MouseSerum

dilutiona

IC50 (ng/well)

SUL(hapten) SPY STZ SDZ SBZ SAA SAM SMZ

1 12 800 119.6 181.3 635.8 60 000b n.i.c n.i. n.i. n.i.2 400 1832.8 n.i. n.i. n.i. n.i. n.i. n.i. n.i.3 12 800 204.3 n.i. n.i. n.i. n.i. n.i. n.i. n.i.4 400 32.5 n.i. n.i. n.i. n.i. n.i. n.i. n.i.5 12 800 n.i. n.d.d n.d. n.d. n.d. n.d. n.d. n.i.

aDilution of serum which gave an OD reading of approximately 0.7 absorbance units.b This value was extrapolated, the percent inhibition of control at 5000 ng/well was 29.3%.c n.i., No inhibition at 5000 ng/well.d n.d., Not done.

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 125

curves for SUL, SPY, SNT and STZ are shown in Figure 2. These curves illustrate the wide

range of recognition Sulfa-1 showed toward the sulfonamides as well as the high

reproducibility of the ELISAs. The results from the cross-reactivity studies of Sulfa-1 using

the cELISA with the panel of sulfonamides are shown in Table 3. The MAb recognized eight

sulfonamide drugs at levels below 1.0 m g/well. Using a limit of detection defined as three

times the variance (3 s ) (Keith et al., 1983), three of these compounds , SNT, SPY and STZ,

should be detected well below the established action level of 0.1 ppm. These results indicated

that the SUL hapten that was synthesized could be used to produce MAbs that cross-reacted

with several sulfonamide drugs that varied in structure at both the N 1 and N 4 positions.

Antibody Sulfa-1 was nearly 50 times more sensitive toward SNT and nearly three times

more sensitive toward SPY than toward the free hapten. SNT, an anticoccidial agent used

in veterinary medicine, is structurally similar to SUL in the center portion of the molecule,

possessing two benzyl ring systems connected by the sulfonamide linkage (Table 1).

However, SNT differs from SUL on the distal ends of the molecule, one end being

N 4-acetylated and the other end possessing a nitro group in place of the carboxyl group

which is found on the free hapten. Mass spectral analysis confirmed the integrity of SNT

( + FAB/MS (M + 1) m/z 336). HPLC analysis, MS and NMR spectroscopy confirmed the

integrity of the hapten, SUL. The structural differences between SNT and SUL were

sufficient to cause more antibody recognition of SNT. In addition, SPY, which differs from

the hapten in the N 1-ring (pyridinyl), and is missing the carboxyl group, also was

recognized by the antibody to a greater extent than SUL. Antibody recognition was

decreased when a thiazolyl ring system was substituted at the N 1 position (STZ) and was

nearly lost when a pyrimidine ring system was substituted (SDZ). This suggested that the

antibody was somewhat tolerant of the presence of a single nitrogen in the N 1-ring

adjacent to the point of attachment (SPY and STZ), but was not tolerant of substituting

FIG. 2. Competitive inhibition ELISA curves for MAb Sulfa-1 with the hapten (SUL), sulfanitran (SNT),sulfapyridine (SPY) and sulfathiazole (STZ). B/B0 = (absorbance of sample/absorbance of control(no competitor)) 3 100. Error bars are the standard deviations from duplicate wells. j , SUL;d , SNT; m , SPY; . , STZ.

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126 M. T. MULDOON ET AL.

two nitrogens in the N 1-ring (SDZ). The five-membered thiazolyl ring system of STZ

contains a sulfur at one of the positions and significant antibody recognition was

maintained. The greater recognition of SIM over SMZ may be accounted for by the

differences of the two pyrimidine ring nitrogens in relation to the point of attachment to

the N 1 position. In addition, the greater recognition of STZ over SMT and SMR over SMZ

suggests that, in general, the presence of N 1-ring alkyl substituents decreased antibody

recognition.

The observation that SNT, an N 4-acetylated sulfonamide, was strongly recognized by the

Sulfa-1 MAb suggested that the hapten may have been N 4-acetylated in vivo, since this is a

common metabolic pathway of sulfonamides in mammals (Lindsay & Blagburn, 1995). If

this were the case, the antibodies that were produced should more readily recognize

N 4-acetylated versions of the competitors. To test this hypothesis, N 4-acetylated SPY

(N 4-ASPY) was synthesized and tested in the cELISA. If the N 4-acetyl group was an

important binding epitope, this compound should have been better recognized than the non-

acetylated form (SPY). However, N 4-ASPY was recognized by the antibody nearly 600 times

less than SPY. The N 4-acetylated SAM (N 4-ASAM) and N 4-acetylated SMZ (N 4-ASMZ)

were tested. Similarly to N 4-ASPY, these also showed very limited recognition by the

antibody (less than 0.5%). Therefore, N 4-acetylation does not explain the antibody

recognition pattern that was observed. The above results suggest that the structural similarity

of SNT to SUL at the N 1-ring and the central region of the molecule are dominant features

contributing to the high degree of antibody recognition for SNT. Consistent with this

conclusion was the lack of recognition of SBM and the low, but detectable recognition of

SSZ that may be attributed to the similarity of this compound to the hapten (and SPY) in the

central region of the molecule. The azo linkage at the N 4 position appeared to have less of

TABLE 3. Cross-reactivity of various sulfonamides with MAb sulfa-1

Competitor IC50 (ng/well) % Reactivity

SNT 0.141 4801.42SPY 2.28 296.93SUL (hapten) 6.77 100.00STZ 32.2 21.02SIM 313.6 2.16SMR 374.3 1.81SSZ 430.5 1.57SCP 436.0 1.55SDZ 976.3 0.69SAT 1434.9 0.47SDM 1572.4 0.43N 4-ASPY 1596.7 0.42N 4-ASAM 2000 0.34SQX 2500 0.27SAM 6000 0.11SMX 6000 0.11SMZ 7000 0.10N 4-ASMZ 7000 0.10SMT 7000 0.10SBM 10 000 0.06SFX 20 000a 0.03SAA n i´

b 0.00

aIC50 value was extrapolated (10000 ng/well resulted in a 45%reduction in antibody binding).

b n.i., No inhibition at 10 000 ng/well. n.d., Not done. The %CV ofthe IC50 value for SPY (23 curves) was 11.5%.

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 127

Molecular Modeling StudiesMolecular modeling studies were conducted in order to better understand the results from the

above cross-reactivity studies since examination of the two-dimensional structural repre-

sentations alone did not lead to an obvious explanation. It is well known that the three-

dimensional structure is important for antibody recognition of low molecular weight haptenic

compounds (Pressman et al., 1948; Karush, 1956). Therefore, minimum energy conforma-

tions were calculated for the free hapten and most of the sulfonamide drugs tested in these

studies.

Figure 3 shows stereoscopic views of the minimum energy conformation of the hapten

(SUL), a second (slightly higher energy) structural conformation of the hapten (SUL-B), and

the minimum energy conformations of three cross-reactive sulfonamide drugs (SNT, SPY,

STZ) depicted as ball and cylinder models. The molecules shown in Figure 3 are all oriented

such that the oxygens of the sulfonamide linkage extend into the page. Examination of these

conformations demonstrated that the minimum energy conformation of the hapten was

substantially different than the minimum energy conformations of the cross-reactive

sulfonamide drugs. Specifically, the dihedral angles 1 and 2 for the minimum energy

conformation of the hapten were ±146.5Ê and 60.0Ê, while those of the cross-reacting drugs

SNT, SPY and STZ were 138.6Ê, 137.2Ê and 140.0Ê, and ±57.4Ê, ±60.1Ê and ±52.1Ê,

respectively. These data suggest that the structural conformation of SNT, SPY and STZ may

be important for antibody recognition. The minimum energy conformation of the hapten

(SUL) linked via a peptide bond to the R group of lysine was calculated in order to mimic

the conjugated form of the hapten (not shown). The conformation of this structure was

similar to the unconjugated form of the hapten (SUL). The pyrimidine sulfonamides, SDZ

and SMR, which showed limited antibody recognition, had minimum energy conformations

similar to the hapten. Dihedral angles 1 and 2 for SDZ and SMR were ±137.6Ê and ±104.1Ê,

and 52.3Ê and 31.4Ê, respectively. However, SMZ and SDM, which also showed limited

antibody recognition, had minimum energy conformations with dihedral angles 1 and 2 that

were similar to the cross-reactive drugs. This result suggested that structural conformation

alone is not the only basis of antibody recognition. But that atomic substitution resulting in

further electronic properties also contribute to antibody binding .

Assuming the minimum energy conformation calculated for the hapten is the most

prevalent conformation presented to the animal immune system for the elicitation of specific

antibodies, most of the antibodies produced should be specific for that conformation.

However, since the monoclonal antibody Sulfa-1 recognized sulfonamide drugs with

minimum energy conformations different than that of the hapten, the hybridom a that was

isolated may have been derived from a splenocyte which was activated by a form of the

antigen in which the hapten was in a different structural conformation (the actual binding

conformation) than the minimum energy conformation calculated for SUL. The conformation

of SNT probably more closely resembles the preferred binding conformation for this

antibody since this structure was recognized to the greatest extent. The second structural

conformation of the hapten shown in Figure 3 (SUL ±B) is a representation of an alternative

conformation for the hapten at a slightly higher potential energy ( D E = + 0.193 kcal mol±1 ).

This higher energy conformation may represent the actual recognized conformation of the

hapten that was bound by a lymphocyte and ultimately resulted in the Sulfa-1 hybridoma that

was isolated. The differences in minimum energy conformations observed for the hapten and

the cross-reactive drugs may account for the following observations. First, most of the

an effect on antibody recognition than did N 4-acetylation (N 4-ASPY). These antibody

characterization studies suggested that, relative to the free hapten, changes in substituents at

the N 4 position, the central region, and in the N 1-ring, all have some effect on antibody

recognition. In addition, a combination of substitutions at these positions appeared to produce

synergistic effects on antibody recognition.

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128 M. T. MULDOON ET AL.

FIG. 3. Calculated minimum energy conformation of the hapten (SUL), sulfanitran (SNT), sulfapyridine(SPY) and sulfathiazole (STZ) and the proposed binding conformation of the hapten (SUL-B) shownas stereoscopic pairs of ball and cylinder models.

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 129

antibodies obtained from immunized mice bound the immobilized hapten but were not

inhibited by the free hapten or free drug. Second, a group of antibodies were inhibited by

only the free hapten (i.e. not by the free drug). Third, the majority of the antibody-secreting

hybridomas were not inhibited by free sulfonamide drug.

Electronic Molecular PropertiesThe electronic properties of the calculated minimum energy conformations also were studied.

Visual depictions of the electron density surface of two conformations of the hapten (SUL

and SUL ±B), SNT, SPY, STZ, and N 4-ASPY are shown in Figure 4. Electrostatic potential

energy surfaces result from coloring the electronic density surfaces with the electrostatic

potential. The electrostatic potential at a point near a molecule is the potential energy of a

proton placed at that point. The light areas indicate positive potential and the dark areas

indicate negative potential. Both the SUL and SNT electrostatic potential surfaces show high

electron density and charge localization corresponding to the carboxyl and nitro groups of

SUL and SNT, respectively. Also, in both com pounds there was charge localization on the

aromatic ring linked to the sulfur. The major electronic difference between these two

compounds is the high electron density and associated negative electrostatic potential with

the acetyl group on SNT. However, the cross-reactivity studies demonstrated that with SNT,

the presence of the acetyl group was not sufficient to decrease antibody binding.

FIG. 4. Calculated Minimum energy conformations of the hapten (SUL), sulfanitran (SNT), sulfapyridine(SPY), N 4-acetylsulfapyridine (N 4-ASPY), sulfathiazole (STZ) and the proposed binding conforma-tion of the hapten (SUL-B) depicted as electron density surfaces colored by electrostatic potential.The electron density probability value used for all calculations was 0.01 electrons ‹±3 . The energyvalues (in atomic units (a.u.)) at each color interface are white-red + 0.09 a.u., red-yellow + 0.03a.u., yellow-green + 0.01 a.u., green-light blue 0.00 a.u., light blue-dark blue ±0.01 a.u., dark blue-purple ±0.03 a.u., purple-black ±0.06 a.u., where 1 a.u. = 627.503 kcal mol±1 .

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130 M. T. MULDOON ET AL.

The molecular depictions of SPY and N 4-ASPY show that they are very similar

electronically in the N 1-ring since they both contain a pyridinyl ring system at that position.

However, they were different in electron density and electrostatic potential on the aromatic

ring linked to the sulfur. Like SNT, the presence of the acetyl group on N 4-ASPY resulted in

a negative electrostatic potential in this region as well as on the adjacent aromatic ring. Also,

it appears that acetylation diminishes the negative electrostatic potential on the part of the

ring opposite the side of the carbonyl oxygen. In regard to SPY, the electronic charge is

evenly distributed on both sides of the ring. These differences in molecular structure and

electronic configuration may be associated with the large differences in antibody recognition

between SPY and N 4-ASPY. The experimental observation suggests that there is an additive

effect concerning the N 1-pyridinyl ring and the N 4-acetyl group that results in the loss of

antibody recognition when both of these groups are present (N 4-ASPY). This does not occur

when only one of these moieties are present (SPY and SNT).

Potential Energy-C onformation MapsThe relative `flexibility’ of the molecules were studied in order to examine the

interchangeability of the various structural conformations and to determine if any differences

in structural or electronic features could explain the recognition pattern that was observed in

regard to SUL, SNT, SPY, and N 4-ASPY. The dihedral angles of the sulfur-ring carbon and

the N 1-nitrogen-ring carbon bonds (dihedral angles 1 and 2, respectively (Figure 3)) appeared

to be important for determining the appropriate binding conformation for the cross-reactive

sulfonamides. Therefore, these two bonds were independently rotated 360Ê in 15Ê increments

(625 com binations) thereby changing the orientation of the two ring systems relative to one

another. The potential energies of each of the resulting conformations were calculated. Figure

5 shows the potential energy map generated for the hapten following this procedure. It can

be seen that the orientation of the two ring systems is highly interdependent. That is, neither

of the bonds can rotate 360Ê independently without reaching prohibitively high potential

energies. However, the potential energy obtained by rotation of one bond can be minimized

by rotation of the other bond. For the hapten, the energy barrier between its calculated

minimum energy conformation (SUL, Figure 3) and the proposed binding conformation

(SUL-B) for Sulfa-1 was 3.13 kcal mol±1 . This energy barrier is most likely accessible at

room temperature, and also further note that the mouse has a body temperature greater than

room temperature. However, to assume the other structure, the dihedral angles have to rotate

simultaneously to maintain the path of minimum energy between these two conformations.

Very similar potential energy maps were obtained for SNT (energy barrier = 2.66 kcal

mol±1 ), SPY (energy barrier = 2.96 kcal mol±1 ), and N 4-ASPY (energy barrier = 2.69 kcal

mol±1 ) between their minimum energy conformations and a conformation similar to SUL.

These data implied that, at room temperature, these compounds probably exist in a

combination of interchangeable conformations, the most prevalent being the global minimum

energy conformation. minimum energy conformation.

The similarities of SNT and N 4-ASPY concerning rotation of dihedral angles 1 and 2

suggested that, in relation to these dihedrals, conformations did not exist for one compound

that were not accessible or interchangeable with the other. Furthermore, no unique

prohibitive conformational energy barrier was observed that could explain the differences in

antibody recognition for these compounds . It cannot be ruled out that antibody binding

involved an antibody-induced conformational change of the drug that resulted in the

preferential binding of SNT. The cross-reactive sulfonamide drugs SPY and STZ have

similar minimum energy conformations as SNT but lack the N 4-acetyl group. Both

compounds fit the antibody binding site, although less tightly, hence the higher IC5 0 values

when compared to SNT. The flexibility of the antibody combining site, facilitating an

`induced fit’ mechanism for antibody-antigen recognition, has been previously described

(Rini et al., 1992). Elucidation of the binding mechanism for Sulfa-1 may be definitively

determined through further spectrom etric and crystallographic studies.

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 131

Kinetic ELISA ExperimentsTo support the hypothesis that antibody recognition may be partially dependant on

sulfonamide structural conformation, cELISA experiments were conducted at both room

temperature (approximately 22ÊC) and 37ÊC. By adding energy to the system, it was possible

that a change in the relative amounts of the various structural conformations would occur for

both the hapten bound to the plate and the free drug present in the sample. The higher

temperature should result in an increase in the amount of antibody bound to the plate if more

BSA ±SUL was in the true binding conformation (SUL ±B). A decrease in antibody

recognition of the cross-reactive drugs SPY and SNT also may have been expected at the

elevated temperature since there should be less SPY and SNT in the appropriate binding

conformation.

Figure 6 shows the results from these experiments. The amount of antibody bound to the

BSA ±SUL-coated plates, when no com petitor was present, expressed as maximum

absorbance, is shown in Figure 6 (top panel). At the early incubation times (between 0.5 and

2 h), there was more antibody bound when the cELISA was conducted at 37ÊC than at room

temperature. At 6 h, antibody binding to the BSA ±SUL appeared to approach equilibrium

and the maximum absorbance at both temperatures were the same. The initial rate of antibody

binding to BSA ±SUL was enhanced at the elevated temperature and this was probably due,

in part, to an increase in the diffusion rate of the antibody in solution.

FIG. 5. Potential energy map of dihedral angles 1 and 2 for the hapten (see Figure 3 for dihedral angledesignations). The highest potential energy is in black, the lowest potential energy is in white.

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132 M. T. MULDOON ET AL.

Antibody recognition of free drug in solution was also time and temperature dependant

(Figure 6 (bottom panel)). At all incubation times, antibody recognition of free sulfonamide

was approximately two times greater at room temperature than at 37ÊC. At both temperatures,

antibody recognition of free sulfonamide decreased over time and this was concurrent with

an increase in maximum antibody binding to BSA ±SUL. This may be interpreted as evidence

that diminished antibody recognition of free sulfonamide was simply a function of the

FIG. 6. Competitive inhibition ELISA conducted at room temperature and 37ÊC. Antibody was incubatedwith the sample on BSA±SUL-coated plates at either room temperature or 37ÊC for either 30 min,1 h, 2 h or 6 h. All other conditions were the same as described for the cELISA. Top panel: Antibodybinding to the BSA ±SUL-coated plate (no competitor present) as measured by the maximumabsorbance. Closed symbols represent experiments conducted at room temperature, open symbolsrepresent experiments conducted at 37ÊC. The error bars represent the standard deviation from themeasurement of 16 wells. Bottom panel: Antibody recognition of free drug as measured by the IC50 .Closed symbols represent experiments conducted at room temperature, open symbols representexperiments conducted at 37ÊC. The competitors were SNT (squares) and SPY (circles). The errorbars represent the standard deviation from IC50 values obtained from triplicate (SNT) or duplicate(SPY) standard curves.

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A CROSS-REACTIVE MAb TO SULFONAMIDE ANTIOBIOTICS 133

antibody reaching equilibrium with BSA ±SUL. However, at 6 h, where the amount of

antibody bound to BSA ±SUL was the same at both temperatures, antibody recognition of

free sulfonamide was still two times greater (lower IC5 0 values) at room temperature than at

37ÊC. This observation may suggest that, at room temperature under conditions approaching

equilibrium, the antibody preferentially recognized the free drug over BSA ±SUL, since more

of the free drug would have been in the appropriate binding conformation. In contrast, at

37ÊC, more BSA ±SUL would have been in the high energy binding conformation while less

of the free drug would probably be in the appropriate low energy binding conformation.

Therefore, the antibody preferentially bound BSA ±SUL, resulting in less recognition (higher

IC5 0 values at 37ÊC) of the free drug in solution.

The cELISA format involves several binding equilibria occurring simultaneously, in

addition to other complex antibody binding interactions, such as induced fit (Rini et al.,1992) and hapten trapping (Schultz & Lerner, 1995). Therefore, other interpretations of the

data may be attempted. However, the data can be explained in terms of temperature-induced

hapten and sulfonamide drug conformational changes. It readily follows from the hapten/free

drug conformational changes as described by molecular modeling, shown by the use of

potential energy-conformational maps, and seen in temperature binding studies, that these

data could indeed explain the antibody hapten/drug binding data observed between SUL,

SPY and SNT with the Sulfa-1 MAb.

CONCLUSIONS

An anti-sulfonamide antibody-secreting hybridoma that cross-reacts with several sulfona-

mides varying in structure at both the N 1 and N 4 position was isolated. This apparently rare

clone was derived from a mouse which was immunized with a N-sulfanilyl-4-aminobenzoic

acid hapten-protein conjugate. Most of the antibodies that were detected in immunized mice

bound either conjugated or free hapten but not relevant sulfonamide drugs. However, through

screening antibody-secreting hybridom as, it was possible to isolate a cell line which

recognized free drug more readily than the hapten. Molecular modeling studies of the hapten

and sulfonamide drugs were conducted in order to better understand the basis for antibody

recognition. These studies showed that the minimum energy conformation of the hapten was

different than those of the cross-reactive sulfonamide drugs. The minimum energy

conformations of the sulfonamide drugs (SPY and SNT) that did bind most effectively were

similar to a higher energy conformation of the hapten. It is suggested that the higher energy

conformation of the hapten could be attained at the body temperature of the immunized mouse.

Thereby, resulting in the production of antibodies to this higher energy conformation of the

hapten. This was supported by evaluating conformations of the hapten and sulfonamides by

molecular modeling, potential energy-conformational maps derived from molecular modeling,

and by ELISA kinetic studies which showed that both antibody binding to immobilized hapten

(BSA ±SUL) and antibody recognition of a free sulfonamide drug (SNT and SPY) in solution

were affected by temperature. Future design of sulfonamide haptens in particular, and other

haptens in general, should consider not only the two-dimensional structure but also the three-

dimensional conformation and various structural com binations that can be easily attained

within the body temperature of the immunized animal.

ACKNOW LEDGEMENTS

Mention of a trade name, proprietary product, or specific equipment does not constitute a

guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval

to the exclusion of other products that may be suitable.

Cartesian co-ordinates for the models can be obtained from Ross C. Beier

(RCBeier@ffsru.tamu.edu).

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